Explanation by a putative triester‐like mechanism for the thio effects and Mn²⁺ rescues in reactions catalyzed by a hammerhead ribozyme

Abstract

Divalent metal ion-dependent hammerhead ribozymes can cleave any RNA with a NUX triplet, wherein the N can be any residue and X can be C, U or A. In recent literature on the mechanism of action of hammerhead ribozymes, one important role of divalent metal ions is generally suggested to be an electrophilic catalyst by directly coordinating with the pro-Rp oxygen of the scissile phosphate to stabilize the transition state. This proposal was made on the basis of thio effects and the proposed electrophilic catalyst is very attractive as an explanation for the catalytic activity of metalloenzymes. Reexamination of thio effects with substrates having a GUA triplet at the cleavage site shows that, in agreement with the previous finding, the cleavage rate, in the presence of Mg P+ ions, is significantly reduced in the case of the phosphorothioate substrate (RpS), wherein the pro-Rp oxygen at the scissile phosphate is replaced by sulfur, while the cleavage rate is reduced to a much lesser extent for the other isomer (SpS), wherein the pro-Sp oxygen at the scissile phosphate is replaced by sulfur. However, more careful examination of the rescue ability of Mn P+ ions with these isomers demonstrates that more thiophilic Mn P+ ions rescue the reaction not only with the RpS isomer but also with the SpS isomer and, importantly, to a greater extent for the SpS isomer. These results argue against the previous conclusion that a metal ion is directly coordinating with the pro-Rp oxygen of the scissile phosphate to stabilize the transition state. In this paper we try to elucidate the possible origin of the thio effects and propose à triester-like' mechanism in reactions catalyzed by hammerhead ribozymes.z 1998 Federation of European Biochemical Societies.

Mapping of the functional phosphate groups in the catalytic core of deoxyribozyme 10–23

“…According to the HSAB (Hard and Soft, Acid and Base) rule [21], a reduction in the cleavage rate of a 'soft' thio-substituted substrate should be observed in the presence of 'hard' Mg 2+ cation (the thio effect), and restoration of a normal cleavage rate of a sulfur-containing substrate should occur in the presence of thiophilic cations such as Mn , in increasing order (the rescue effect). Analysis of these types of interaction led to a better understanding of the mechanistic aspects of the action of naturally occurring catalytic ribozymes: group I and II introns [22][23][24][25], the RNA subunit of RNase P [26,27], and the hammerhead ribozymes [18,[28][29][30]. The successful application of Pchiral phosphorothioates in those mechanistic studies prompted us to establish the role of phosphate groups in the catalytic core of deoxyribozyme [10][11][12][13][14][15][16][17][18][19][20][21][22][23].…”

CHARMM force field parameters for simulation of reactive intermediates in native and thio‐substituted ribozymes

“…77 If the divalent metal ion binding at a certain position is required for catalysis, a large decrease in the reaction rate should be seen upon thio substitution at this position. However, the interpretation of thio affect results are sometimes inconclusive 21,23,78,79 especially when possible conformational changes induced by the sulfur are considered. Molecular simulation of thio-substituted phosphates and phosphoranes and their role on structure would aid in the interpretation of experimental thio effects.…”

The effect of divalent cations on the catalytic activity of the human plasma 3′-exonuclease

“…, or Cd 2? (the rescue effect), P-chiral phosphorothioates have been widely used in numerous mechanistic studies (Warnecke et al 1996;Zhou et al 1998;Wang et al 1999). In this work we have shown that the cleavage efficiency of the PS-oligo 2 in HP containig Mg 2?…”